Inhibitory Effect Of HDACi Valproic Acid Combined With Ionizing Radiation On The Proliferation Of Breast Cancer Cells And Its Mechanisms

Background:Breast Cancer（BC）is the most common cancer and the second leading cause of cancer-related deaths.Due to resistance of breast cancer targeted drugs and triple-negative breast cancers（TNBC）have no obvious tumor-specific receptors or targeted pathways,finding new drug targets in breast cancer has Important clinical significance.Histone deacetylase inhibitors（HDACi）are a new type of small molecule therapeutic drugs,which show great promise in tumor treatment.Valproic acid（VPA）is a type I HDACi,which has received widespread attention in cancer epigenetics research.VPA causes high acetylation of the N-terminal tails of H3 and H4 in vitro and in vivo,and inhibits histone deacetylase（HDAC）activity by binding to the active center of the enzyme and blocking the entry of the substrate.Like other HDACi,it is a non-cytotoxic anti-tumor drug.Its mechanism of action is mainly to change the transcription level of different genes by changing the histone acetylation status of tumor cells,thereby causing a wide range of anti-tumor effects,including inhibition A variety of tumor cell proliferation,promote differentiation,induce apoptosis,enhance the sensitivity of chemotherapy and radiotherapy,and reverse tumor metastasis.Purpose:To study the effect of VPA alone or combined with X-ray irradiation on the proliferation inhibition of breast cancer MCF-7（p53 wild type）and MDA-MB-231（p53 mutant）cells,and to explore the effects of VPA alone or combined with X-ray irradiation on cell cycle,apoptosis,autophagy and repair of DNA damage in two breast cancers.Methods:1.The CCK-8 method was used to detect the cell proliferation activity of different concentrations of VPA alone or combined with X-ray irradiation in MCF-7and MDA-MB-231 cells for 24,48 and 72 h.2.The PI staining method was used to detect the cycle arrest distribution of MCF-7 and MDA-MB-231 cells after VPA alone or combined with X-ray irradiation for 24 h.3.The Annexin V-FICT method was used to detect the apoptosis rate of MCF-7and MDA-MB-231 cells after VPA alone or combined with X-ray irradiation for 24and 48 h.4.The MDC staining method was used to detect the autophagy rate of MCF-7and MDA-MB-231 cells after VPA alone or combined with X-ray irradiation for 24and 48 h.5.The Rh123 staining method was used to detect the changes in mitochondrial membrane potential of MCF-7 and MDA-MB-231 cells after VPA alone or combined with X-ray irradiation for 24 and 48 h.6.Real-time fluorescent quantitative PCR was used to detect the m RNA expression levels of genes related to cell cycle,DNA damage,apoptosis,and autophagy in MCF-7 and MDA-MB-231 cells after VPA alone or combined with X-ray irradiation for 24 and 48 h.7.Western blotting was used to detect the protein expression levels of HDAC2and LC3A in MCF-7 and MDA-MB-231 cells after VPA alone or combined with X-ray irradiation for 24 and 48 h.8.The experimental results are statistically analyzed using SPSS 24.0 software.The experimental data are expressed in terms of mean deviation±standard deviation（x±s）.The sample mean between multiple groups is analyzed by one-way analysis of variance,and the pairwise comparison between groups is performed by SNK test.P<0.05 indicates that the difference is statistically significant.Results:1.The effect of VPA combined with X-ray irradiation on the proliferation activity of MCF-7 and MDA-MB-231cellsAfter different concentrations of VPA alone or combined with 4 Gy X-ray irradiation on MCF-7 and MDA-MB-231 cells for 24,48 and 72 h,the cellproliferation rate of the VPA group and VPA combined with 4 Gy X-ray irradiation group were significantly reduced（P<0.01）,the inhibitory effect of VPA combined with X-ray irradiation on the proliferation of MDA-MB-231 cells was better than that of VPA alone or X-ray irradiation（P<0.05）.2.The effect of VPA combined with X-ray irradiation on the apoptosis rate of MCF-7 and MDA-MB-231 cellsAfter VPA alone or combined with 4 Gy X-ray irradiation in MCF-7 and MDA-MB-231 cells for 24 and 48 h,the apoptosis rate of VPA group,X-ray irradiation group and VPA combined X-ray irradiation group increased significantly（P<0.05 or P<0.01）,and the apoptosis rate of VPA combined with X-ray irradiation was significantly higher than that of X-ray alone（P<0.05）.3.The effect of VPA combined with X-ray irradiation on the autophagy rate of MCF-7 and MDA-MB-231 cellsAfter VPA alone or combined with 4 Gy X-ray irradiation on MCF-7 cells for 24and 48 h,the autophagy rate of X-ray irradiation group,VPA group and VPA combined X-ray irradiation group were significantly reduced（P<0.05）.After VPA alone or in combination with 4 Gy X-ray irradiation on MDA-MB-231 cells for 24 and 48 h,the autophagy rate of X-ray irradiation group,VPA group and VPA combined X-ray irradiation group increased significantly（P<0.01）.After 48 hours of treatment,the autophagy rate of the VPA combined with X-ray irradiation group was higher than that of the VPA group（P<0.05）.4.The effect of VPA combined with X-ray irradiation on the cell cycle of MCF-7 and MDA-MB-231 cellsAfter VPA alone or combined with 4 Gy X-ray irradiation in MCF-7 and MDA-MB-231 cells for 24 h,VPA combined with X-ray irradiation caused G₁phase and G₂/M phase arrest in MCF-7 and MDA-MB-231 cells.5.The effect of VPA combined with X-ray irradiation on cell membrane potential of MCF-7 and MDA-MB-231 cellsVPA alone or combined with 4 Gy X-ray irradiation in MCF-7 and MDA-MB-231 cells for 24 and 48 h.In MCF-7 cells,the mitochondrial membrane potential of X-ray irradiation group,VPA group and VPA combined X-ray irradiation group All were significantly reduced（P<0.05）;in MDA-MB-231 cells,all groups were significantly increased（P<0.05）.6.Study on the mechanism of VPA combined with X-ray irradiation in MCF-7 and MDA-MB-231 cellsAfter VPA alone or combined with 4 Gy X-ray irradiation in MCF-7 and MDA-MB-231 cells for 24 and 48 h,in MCF-7 cells,the expression of p53,mTOR,Beclin-1,p21 and LC3 m RNA were increased（P<0.05 or P<0.01）,and the expression of ATM,EGFR,NFκB1 and RAD51 m RNA were decreased（P<0.05 or P<0.01）in the VPA combined with X-ray irradiation group.In MDA-MB-231 cells,the expression of p53,mTOR,Beclin-1,EGFR,NFκB1,RAD51,p21 and LC3m RNA were increased（P<0.05 or P<0.01）,and the expression of ATM m RNA was decreased（P<0.01）in the VPA combined with X-ray irradiation group.After VPA alone or combined with 4 Gy X-ray irradiation in MCF-7 cells,the expression of HDAC2 was decreased,and the expression of LC3A wad increased in the VPA combined X-ray irradiation group.After VPA alone or combined with 4 Gy X-ray irradiation in MDA-MB-231 cells,the expression of HDAC2 and LC3Awere decreased in the VPAcombined with X-ray irradiation group.Conclusions:1.VPA inhibits the proliferation of breast cancer MDA-MB-231 cells by inducing apoptosis,autophagy and causing G₂/M phase arrest;it inhibits the proliferation of MCF-7 cells by inducing apoptosis,repairing DNA damage and causing G₁phase arrest.2.VPA alone or combined with X-ray irradiation can effectively inhibit the proliferation activity of breast cancer MDA-MB-231 cells,and the proliferation inhibitory effect of VPA combined with X-ray irradiation is better than VPA alone or X-ray irradiation alone.The mechanism is that VPA combined with X-ray irradiation causes G₂/M phase arrest of MDA-MB-231 cells;and it induces and promotes autophagy to drive cell death.3.The inhibitory effect of VPA combined with X-ray irradiation is better than VPA alone or X-ray alone does not appear in breast cancer MCF-7 cells,the reason may be that the expression levels of HDAC2 proteins and p53 m RNAin the two cells are different.